Purification of viruses on treated calcium dihydrogen orthophosphate monohydrate adsorbent

ABSTRACT

This invention relates to an alkali treated calcium dihydrogen orthophosphate monohydrate adsorbent. Further, it concerns an alkali and trimetaphosphate treated calcium dihydrogen orthophosphate monohydrate adsorbent. Still further, it relates to the processes for the preparation of these adsorbents and to their use in virus purification.

United States Patent l l3,622,663

[72] Inventor Milton Lapidus [52] .800 424/89 Rosemom, PL 95/1; [211 P 10,644 7o 511 161.01 ..A6lr27/00, [221 Wed B0ljll/82,C01b25/32 [451 paemd 1971 s01 FieldolSearch 424/89- [73] Assignee American HomeProducts Corporation New York, N.Y. 195/ l Original application June 13, 1968, Ser. No. 736,561, now Patent No. 3,509,070, which is a continuation-in-part of application Ser. No. 497,540, Oct. 18, 1965, now abandoned which is a continuation-in-part of application Ser. No. 396,726, Sept. 15, 1964, now abandoned. Divided and this application Feb. 11, 1970, Ser. No. 10,644

[54] PURIFICATION OF VIRUSES 0N TREATED CALCIUM DIHYDROGEN ORTHOPHOSPHATE MONOHYDRATE ADSORBENT 5 Claims, No Drawings Primary Examiner-Richard L. Huff Attorneys-John J. Hagan, Joseph Martin Weigman and Edmund H. OBrien ABSTRACT: This invention relates to an alkali treated calcium dihydrogen orthophosphate monohydrate adsorbent. Further, it concerns an alkali and trimetaphosphate treated calcium dihydrogen orthophosphate monohydrate adsorbent. Still further, it relates to the processes for the preparation of these adsorbents and to their use in virus purification.

PURIFICATION OF VIRUSES N TREATED CALCIUM DIHYDROGEN ORTHOPHOSPHATE MONOHYDRATE ADSORBEN'I This application is a division of application Ser. No. 736,561, filed June 13, 1968, now U.S. Pat. No. 3,509,070, which is a continuation-in-part of copending US. Application, Ser. No. 497,540, filed Oct. 18, 1965 and now abandoned, which is a continuation-in-part of its parent US. Pat. application, Ser. No. 396,726, filed on Sept. 15, 1964, which is now abandoned.

This invention relates to the purification and concentration of virus. More particularly, this invention relates to new and improved adsorbent compositions particularly effective in purifying influenza virus. The present invention also encompassed the novel method by which these new adsorbent compositions are prepared as well as the method by which the same can be utilized in preparing highly purified forms of influenza virus.

in the US. Pat. No. 3,368,867, entitled, Chromatographic Purification of Influenza Virus With Brushite Modified by Autoclaving," filed on Sept. 15, 1964 and issued on Feb. 13, 1968, there is described a new adsorbent of the calcium phosphate family, namely, autoclaved brushite. As described therein, it was found quite unexpectedly that by autoclaving brushite, separation and concentration of influenza virus from allantoic fluids containing the same could be substantially improved. It is further shown in US. Pat. No. 3,368,867 that the autoclaved brushite made possible a substantial improvement in the degree or level of purification of Sharples concentrated B strain influenza virus.

It has now been discovered according to the present invention that another calcium salt, namely, calcium dihydrogen orthophosphate monohydrate [Ca( H,PO,),-H,O] if treated according to the method of the present invention is effective in separating various strains of virus including the B strain from influenza infected allantoic fluid. it has also been found that the effectiveness of this adsorbent composition is further improved if the adsorbent is treated with an alkali metal trimetaphosphate e.g. sodium trimetaphosphate or potassium trimetaphosphate prior to use. Utilization of an alkali metal trimetaphosphate for this purpose as distinguished from the conventional phosphate buffers such as dibasic sodium phosphate (Na,HPO,) affords an unexpected increase in recovery of A strains of influenza virus and an increased purification factor, generally in the order of four to five times.

In addition and more importantly, the new adsorbent of the present invention when treated with an alkali metal trimetaphosphate substantially improves both the yield and purification of B strains of influenza virus from the fluids in which such strains are normally present. This improvement is in the order of a four to five fold increase in recovery and up to a -fold increase in the degree or level of virus purification obtained. This and other advantages are provided by the novel adsorbent of the present invention and the method in which the same is used.

Considered in its broadest aspect, the new adsorbent of the present invention is the monohydrate of calcium dihydrogen orthophosphate which is treated with an aqueous solution of an alkali to provide a suspension having a pH in the range of about 5.5 to about 8.0, preferably about 6.5. As employed herein the tenn alkali is meant to comprise those aqueous solutions which contain compounds which yield hydroxyl ions upon dissolution. Many such alkali will readily suggest themselves to those skilled in the chemical art. Some examples thereof are: sodium or potassium nitrate, sodium or potassium acetate and sodium or potassium hydroxide. In a preferred embodiment of this invention, calcium dihydrogen orthophosphate monohydrate be mixed with a limited quantity of water such as from about one to not more than about five parts of water per part of salt and preferably from about three to about five parts of water per part of salt should be used. After addition of the monohydrate of calcium dihydrogen orthophosphate, stirring of the resulting suspension is continued during which the alkali is added slowly over a period of from about one-half to about 2 hours until the adjusted pH of about 5.5 to about 8.0 is achieved, preferably abut 6.5. During this period the reaction mixture is kept at a temperature of not more than about 40 C. and preferably a temperature of from about 20 to 30+ C., although temperatures as low as about 0 C. may be employed. Thereafter, the alkali treated adsorbent is permitted to settle and the supernatant liquid is decanted. The residue is then washed with water until the pH of the water wash is at least 0.2 units higher than the adjusted pH of the above suspension and is in the range of from about 5.7 to about 8.2. The alkali treated calcium dihydrogen orthophosphate monohydrate adsorbent may then be kept in the supernatant fluid or the latter can be decanted to leave the solid adsorbent in a stable form which is ready for use. When the alkali treated adsorbent is used for virus purification, the procedure employed is similar to that hereinafter described for the alkali and the TMP treated adsorbent except that the adsorbent is not treated with an alkali metal trimetaphosphate. Further, the virus containing fluid may or may not be TMP treated.

As previously indicated, the preparation of the alkali treated calcium dihydrogen orthophosphate monohydrate adsorbent according to the method described above provides an unexpectedly effective means for recovering a highly purified virus from influenza infected source materials. However, it is an added feature of the present invention to increase and further activate this alkali treated adsorbent by treating the same with sufiicient amount of an alkali metal trimetaphosphate (TMP) preferably sodium trimetaphosphate (Na P O,) until the pH of the adsorbent reached a range from about 7.5 to about 8.5 preferably about 8.0. At this pH, the alkali and TMP treated adsorbent is particularly effective, as is demonstrated more clearly hereinafter, in separating influenza virus from allantoic fluid. This alkali and TMP adsorbent is particularly effective in recovering the B/GL strain of influenza virus fluids containing the same.

When the alkali and TMP treated calcium dihydrogen orthophosphate monohydrate adsorbent is used for virus purification, it is placed or packed in a conventional chromato-- graphic column having the necessary adsorbent support. If the pH of the adsorbent has not previously been adjusted to the desired Ph, e.g. a pH of about 8.0, the same is washed with an aqueous alkali metal trimetaphosphate solution, e.g. sodium trimetaphosphate preferably at a molar content of about 0.01. The virus-containing fluid such as allantoic fluid is usually diluted from about a 2:1 to about a 5:! ratio with water and adjust to a pH range of about 7.0 to about 9.0 after the addition of the alkali metal trimetaphosphate. While effective purification of the virus can be obtained at these dilution and pH ranges, it has been found that higher flow rates are obtained in the column at a preferred dilution ratio of about 2:1 and a pH of about 8.

To concentrate the influenza virus, the alkali metal trimetaphosphate treated and pH-adjusted allantoic fluid containing the virus is then poured onto and through the prepared column. The amount of virus solution delivered to the column will depend on the size of the column and the quantity of adsorbent contained therein. It is preferred that the column be operated in a continuous manner and not permitted to run dry. When the total amount of infected allantoic fluid has been placed on the column, the virus is washed with an aqueous alkali metal trimetaphosphate solution (pH 8), e.g. sodium trimetaphosphate. Thereafter, the virus is recovered by elution. in carrying out this elution to provide a purified concentrate of the virus a conventional buffer with a pH in the range of about 5.0 to about 9.0 having a molar concentration of about 0.3M to about 2.0 M is employed. Preferably a 1.0 M phosphate buffer e.g. dibasic sodium phosphate having a pH of about 6.5 to about 7.0 is used.

Reference now to the specific examples which follow will provide a better understanding of the nature and characteristics of the new adsorbent composition of the present invention, the method by which the same is prepared and the manner in which it is used.

EXAMPLE! Fifty grams of calcium dihydrogen orthophosphate monohydrate [Ca(H,PO,),'H,O] and 200 ml. of distilled water are stirred vigorously for 15 minutes. With stirring a dropwise over a period of one-half hour until a pH of 6.5 is reached. The contents of the reaction mixture is kept at 20- 23 C. After stirring for one hour, the adsorbent is permitted to settle and the supernatant liquid is decanted. The adsorbent 5 is washed batchwise with one liter portions of distilled water 321$ f m until the pH of the supernatant liquid is 7.2-7.4. The adsor- H 2. ed Th d ed bent is then batch washed with a 1.01 M sodium 2:?" 2: 5 12:3: gsgzpgg trimetaphosphate (pH 8) solution until a pH of 8 is reached. mm is removed y dccanmion- The adwrbcm is washed with 10 pH History of Adsorbent During Alkali and TMP Treatment one liter portions of distilled water until the supernatant liquid Cycle has a pH of 7.2-7.4.

pH History of Adsorbent During the Alkali Treatment Cycle 15 'so cr(ri.r0.),-i-i,o'+2oo ml. an; 2.5 109 ml. 3N sodium hydroxide (4' C.) 6.5 cnnmom pH 1 hour ltirring 6.5 I liter water wash [6.8 so ce ri,ro, ,-ii,o+zoo ml. water 2.3 1 i I09 ll. 3N roaitnn hydroxide as i l hour "in," 6,5 l liter 0.01 M, us. sodium tnrnctaphoaphale wash 7.65 1 limwl er Wflh 5,3 l liter 0.01 M. pHB, sodium trimetapheaphate \vuh 8.00 l liter water wash 7.05

1 liter water wash 7.3

Similarly, with like results the above procedure is con- EXAMPLE ll ducted with a 0.01 M potassium trimetaphosphate solution.

One hundred grams of calcium dihydrogen orthophosphate EXAMPLE V monohydrate [Ca(H,PO ),'H,O] and 500 ml. of distilled water are stirred for a half hour. Thereafter, with continuous The treated 'dsmbcms f cxamples m are stirring at room temperature 2N potassium hydroxide is added 'F an aqueous solutfon of mew dropwise over a period of 2 hours until a pH of 6.5 is obtained. mmetaphosphm? f demonmawd examplc w P" The stirring is cominucd for another hour, then the superna of the adsorbent IS in the range of about 7.5 to about 8.5. tant liquid is decanted and the solid residue washed with two liter portions of distilled water until pH of the supernatant EXAMPLE v] liquid is 7.0. Thereafler, the supernatant liquid is decanted to 3 5 Fifty grams of Ca(H,PO ),-H,O treated according to examafford the stable adsorbent. 1e [V is laced in a as column containing a sintered glass P P In like manner. a ratio of calcium y wssi @PL'QEEBEE'E be ief 1299: i EELIQL SEl BE t e",

Lowry Volume, protein, GOA/mg. Percent Sample ml. CAA mg. L.P. P.F. recovery Infected allsntoic fluid 5,000 19,000 925 20. 5 Column Fraction 1 5, 200 54 Column Fraction 2 100 12, 800 15 863. 3 41. 6 67 Now: CCA means chick-cell agglutination units and RF. means purification factor.

EXAMPLE lll Repeating the procedure of example i and ii a mixture containing 1:2 ratio of calcium dihydrogen orthophosphate monohydrate and water is treated with an aqueous sodium bicarbonate solution at 5 C. until the pH of the mixture is 5.5.

' Thereafter, the supernatant liquid is removed and the residue washed three times with two liter portions of water until the pH is in the range of5.7 to 8.2.

The above procedure is repeated with similar results using is delivered 5 liters of diluted infected allantoic fluid containing B/GL virus. The fluid having an adjusted pH of 9.0 contained 0.01 M sodium trimetaphosphate flow through the column over a period of 2 hours. The column is then washed with 200 ml. of 0.01 M sodium trimetaphosphate (pH 8) solution and the virus eluted with 0.5 M phosphate buffer (pH 6.8) solution. in the data reported below, Column fraction 2 represents the recovered virus.

By the above procedure a recovery of 67 percent of the 8 strain is obtained at a purification factor of 4i .6.

Similar results are afi'orded by the alkali and TMP treated adsorbents described in example V when the virus-containing fluid is diluted from about a 2:1 to about 5:! ratio with water, adjusted to a pH range of about 7.0 to about 9.0 and eluted the following alkali agents: sodium carbonate, potassium carf h column with a 0,3 M to a 2.0 M phosphate buffer Lowr bonate, sodium nitrate, potassium nitrate, sodium acetate and potassium acetate.

EXAMPLE IV Fifty grams of calcium dihydrogen orthophosphate monohydrate and 200 nil. of distilled water are stirred vigorously for IS minutes. Under conditions of continuous mixing, a cold (4' C.) 3N sodium hydroxide solution is added having a pH of about 5.0 to about 9.0.

EXAMPLE Vll In a similar manner to example Vl, the purification of influenza virus A/Jap. 305 is carried out on 300 g. of alkali and TMP treated calcium dihydrogen orthophosphate monohydrate adsorbent. The virus is first adjusted to a pH of 9.0. The following results were obtained:

By the above procedure, a recovery of lll percent of the virus is obtained at a purification factor of 47.6.

EXAMPLE VIII In a manner similar to the examples VI and VII, a series of runs are carried out on a variety of influenza virus strains, identified below according to the conditions and with the alkali and TMP treated adsorbent of the present invention. The

columns used have varying capacities of from 46 to 102.1. The following table summarizes the results obtained with these various strains of influenza:

Total CCA Percent CCA/mg. recovered recovery RF. 11.19..

3, 949, 920 79 43 4, 028 5, 904, 101 31 3, 463 A/Ann .Arbon 15, 933, 135 87 28 5, 239 6, 186, 67 32 3, 179 608, 86 38 4, 560 6, 711, 390 102 25 1, 901

1 Based on s ecific activity before and after purification.

S.A.=CCA mg. Lowry Protein.

By the above procedure, recovery of the various strains of influenza viruses varied from 67 percent to l02 percent at purification factors from to 43.

Similar results are obtained when the alkali and TMP' treated adsorbents of example V are employed.

EXAMPLE IX Virus Jan 3 5 PR-B Ann Arbor B/GL Put on Col. CCA 122,000 130,000 144,000 72,000

Recovered CCA.. 67,000 $2,000 as, 200 10, 200

Percent recovery 46. 7 63. 0 26. 4 14. 1

Purilltlon factor- 12. 3 20. 1 7. 2 4. 8

Infected allantoic fluid.

By the above procedure, recovery of various strains of influenza virus varied from 14 percent to 46.7 at purification factors of from 4.8 to 20.1. As will be noted, particularly with respect to the recovery of the B strain Great Lakes (B/Gl) a recovery of fourteen percent is attained at a purification factor of 4.8 with this alkali treated adsorbent which contrasts with the results of example VI in which the alkali and TMP treated adsorbent affords a recovery of 67 percent with a purification factor of M6. in a similar manner the results obtained when purifying A strain/lap. 305 without an alkali metal trimetaphosphate treatment provides a 46.7 recovery and a purification factor of l2.3, whereas when the adsorbent is treated with an alkali metal trimetaphosphate the recovery is 101 percent obtained at a purification factor of 3 1.

Similar results are obtained when alkali treated calcium dihydrogen orthophosphate monohydrate adsorbents of examples II and III are employed to purify the influenza virus strains reported in the prior example.

EXAMPLE X Two columns (Columns A and B) are prepared: for each, 100 grams of CA(H,PO ),I-I O are prepared according to example IV and placed in a glass column. To each column there is then delivered one liter of undiluted infected allantoic fluid containing A/Jap/ l 70 virus which are previously adjusted with 0.01 M sodium trimetaphosphate. For Column A the fluid is preheated to 37' C. while for Column 8 the fluid is cooled to 10 C. Prior to application to the column. For both columns, recoveries by elution with 1M pH 6.8 phosphate buffer are as follows:

Volume. Total Recovery,

Sample ml. CCA percent Infected fluid 1, 000 273, 000 Column A:

Fraction 1 1, 210 4, 840 1. 7 Fraction 2 23 93, 311 34. 2 Column B:

Fraction 1 1, 250 5, 000 2 Fraction 2 27 113, 292 44. 5

It should be noted that in both columns above improvement in the concentration of CCA units per ml. is achieved from the starting level of 273 CCA per ml. in the allantoic fluid. The concentration is approximately l5-fold for both columns (4057 CCA per ml. in A and 4196 CCA per ml. in B).

EXAMPLE X! In a manner comparable to the preceding example, 750 ml. of A2/Jap/l70 virus are applied to the column at a temperature of 4' C. after having been diluted with an equal volume of water and then adjusted to 0.01 M sodium trimetaphosphate. The recovery of virus so processed is as follows:

V olume, Total Recovery Sample ml. CCA percen Infected fluid 1, 600 64, 500

Fraction 1 1, 680 6, 720 1. 7

Fraction 2 29 40, 136 62 EXAMPLE XII In a manner similar to the preceding examples, a column is prepared in which cold 3 N sodium hydroxide is added dropwise to the adsorbent until a pH of 3.0 is reached. The recovery of A/Jap/ l 70 virus so processed is as follows:

Total Percent Two columns are prepared: for each, l00 grams of Ca(I-I2PO4)2 H2O are prepared according to example IV. After adjusting to pH 8.0 with 0.01 Molar sodium timetaphosphate buffer wash, the adsorbents are maintained in the buffer wash overnight at room temperature. The adsorbents are then placed in glass columns and adjusted to pH 7.0 (Column A) and pH 7.5 (Column B. with 0.01 Molar sodium trimetaphosphate bufi'er wash. A volume of 500 ml. of B/Maryland strain infected allantoic fluid is applied to each column, after having been diluted with an equal volume of distilled water and adjusted to pH 8.0 with a 0.01 Molar sodium trimetaphosphate solution. The virus is recovered from the columns by elution with 1.0 Molar phosp ha twifer. The following results are obtained:

Volume, CCA/ Total Percent Sample ml. ml. CCA recovery Infected allantoic fluld- 500 217 108, 500

Column A, Fraction 2 67 1,469 98,423 91 Column B, Fraction 2 70 1, 204 84, 280 77 EXAMPLE XIV phosphate buffer, and the following results are obtained:

Volume, CCA] Total Percent Sample ml. ml. CCA recovery Infected allantoic fluids. 750 289 216, 750 Fraction I 1,815 41 74,415 34 Fraction 2 44 976 42. 944 20 It should be noted that concentration and recovery with a TM? treated column having a pH of 9.0 is not as good as that obtained at a TM? column pH of 7.0 and 7.5 respectively, as shown in example XIII. However, some improvement in the concentration of CCA units per ml. is achieved with a 5 trimetaphosphate treated column having a pH of 9.0.

EXAMPLE XVIII In a manner similar to the preceding examples, the purification of A/Jap 170 strain of influenza virus is carried out on eight columns. The virus is eluted with 1.0 Molar phosphate buffer at pH ranges from 5.0 to 9.0."Tha following results are obtained:

Elutlon, Volume, CCAI Total Percent Sample pH ml. ml. CCA recovery Infected nllantoic fluid 750 124 93, 000 C man A 6. 8 32 2, 104 67, 328 72 Column 13 7. 5 33 1, 742 I7, 486 02 Column C 8. 33 1. 375 45. 375 49 Column D 9. 0 59 826 48, 134 52 Infected allantolc fluld. 750 1 232 000 umn E 5. 0 30 3, 705 111,150 64 Column F-.- 5. 34 4, 728 100, 762 92 Column 6. 0 4, 181, 740 104 Column H s. 6 36 4, 885 m, an 101 EXAMPLE XV EXAMPLE XIX In a manner similar to example IV, the purification of B/Maryland strain of influenza virus is carried out on four 20 columns containing 100 grams of Ca(H,PO,),-I-I,O per column. The virus is eluted with LG Molar phosphate buffer, pH 6.8, 7.5, 8.0 and 9.0 respectively. The following results are In a manner similar to the preceding examples, the purification of A lTaiwan strain of influenza virus is carried out on six columns. The virus is eluted with 1.0 Molar phosphate buffer at pH ranges from 5.0 to 8.0. The following results are obq ineq= 9 122, 1

Elutlon, Volume, CCA Total Percent Sample pH ml. ml. CCA recovery Infected allantoie fluids. i m0 427 213, 500 Col A 6. 8 38 4, 595 174, 610 82 7. 5 42 5. 153 210, 426 101 8. 0 42 5, 302 222, 624 104 Column D 9. 0 53 4, 782 253, (46 119 Elutlon, Volume, CCA/ Total Percent Sample pH in ml. CCA recovery Infested allantele fluid 750 295 221, 2!) Column 6. 0 4, 93s 511 89 Column I 0. 5 34 4,! 108,070 74 Column C 7. 6 38 4, 137 I67, I) 71 Column D 8.0 43 4, N0 159, 72 Infected allantolc fluid. 750 205 154, 500 011111111 E 6. 6 46 2, 831 1!), 226 84 Column 1 0. 0 38 2, 391 90, 8

EXAMPLE XVI 4o EXAMPLE xx In a manner similar to example XV, the purification of AJAA strain of influenza virus is carried otlt on four columns. The virus is eluted with 1.0 Molar pheaphate buler, pH 6.8, 7.5, 8.0 and 9.0. The following results were obtained:

In a manner imilar to the preceding examples, the purification of A/Jap 170 strain of influenza virus is carried out on a series of columns. The undiluted virus fluid is applied to the columns and is eluted with phosphate buffer, pH 6.8 and 8.0 at

Elutlon Volume, OCA/ Total Percent Sample pli ml. ml. CCA recovery I Infected allantolc fluld 750 201 160, 750 Column 6. 8 21 6, 054 100, 134 71 Column B 7. 5 38 2, 624 99, 712 66 Column C 8. 0 43 8, 258 140, 094 93 Column D. 9. 0 37 2, 479 91, 723 61 EXAMPLE XVII 0.5 M, 0.75 M, and 1.0 M concentrations. Recoveries of the In a manner similar to the preceding examples, the purifica- Virus Pr ss d are as follows:

tion of A/PRJ strain of influenza virus is carried out on four columns. The virus is eluted with 1.0 Molar phosphate buffer, p32,, and 9.0. The following results are obtained:

7 While the foregoing invention has been described with some degree of particularity in the descriptive matter and in the specific examples provided, the same was for purposes of Elutlen Votnme, CCA/ Total Percent Sample p ml. ml. GOA recovery Infected allnutolc fluids 750 329 m, 760

clarity and definition and is not to be considered in any way as a limit on the scope of the invention. The invention is to be limited only by the claims appended hereto.

What is claimed is:

l. A method of purifying influenza virus which comprises packing a chromatographic column with calcium dihydrogen orthophosphate monohydrate, treating said column at a temperature of not more than about 40 C. with alkali so as to adjust pH to from about 5.5 to about 8.0, then washing said column with water until the pH thereof is raised at least about 0.2 units and is in the range of from about 5.7 to about 8.2, washing said column with an alkali metal trimetaphosphate solution, introducing to the column a virus containing fluid, said virus having been treated with an alkali metal trimetaphosphate at pH of about 7.0 to about 9.0 and eluting said column with a buffer having a pH of about 5.0 to about 9.0.

2. A method as described in claim 1 for purifying influenza virus which comprises packing a chromatographic column with calcium dihydrogen orthophosphate monohydrate, treating said column with sufficient alkali to adjust its pH to the range of from about 5.5 to about 8.0 at a temperature of not more than about 40 C., said column thereafter being washed with sufficient water to raise the pH of the column at least about 0.2 units to the range of about 5.7 to about 8.2, further washing said column with an alkali metal trimetaphosphate solution having a pH of about 8, flowing unto the column a diluted virus solution, said virus solution having been treated with an alkali metal trimetaphosphate and having a pH of about 8.0 and eluting said column with a phosphate buffer having a pH of about 7.0.

3. A method as described in claim I for purifying influenza virus which comprises packing a chromatographic column with calcium dihydrogen orthophosphate monohydrate, treating said column with sufficient alkali to adjust its pH from about 5.5 to about 8.0 at a temperature of not more than about 40 C., said column thereafter being washed with sufficient distilled water to provide the column with a pH of about 7.5, further washing said column with a solution of an alkali metal trimetaphosphate, flowing unto the column a diluted influenza virus containing fluid, said virus having been treated with an alkali metal trimetaphosphate so as to have a pH of about 8.0 and eluting the influenza virus from said column with a phosphate buffer having a molarity of about 0.3 to about 1.0 and a pH of about 7.0.

4. A method as described in claim 1 for purifying influenza virus which comprises packing a chromatographic column with calcium dihydrogen orthophosphate monohydrate, treating said column at about room temperature with sufficient alkali to adjust the pH to about 6.5, said column thereafter being washed with sufficient distilled water to provide the column with a pH of about 7.5, further washing said column with a sodium trimetaphosphate solution having a pH of about 8, flowing unto the column a dilute influenza virus containing fluid, said virus having been treated with sodium trimetaphosphate so as to have a pH of about 8.0 and eluting the influenza virus from said column with a LG molar phosphate buffer having a pH of about 7.0.

5. A method for purifying virus which comprises packing a chromatographic column with calcium dihydrogen orthophosphate monohydrate, treating said column at a temperature of not more than about 40 C. with sufficient alkali to adjust its pH to the range of from about 5.5 to about 8.0, said column thereafter being washed with sufficient water to raise the pH of the column at least about 0.2 units to the range of about 5.7 to about 8.2, further washing said column with a solution of an alkali metal trimetaphosphate, flowing unto the column an influenza virus containing fluid and eluting said column with a phosphate buffer having a pH of about 7.0.

i i i i i 

2. A method as described in claim 1 for purifying influenza virus which comprises packing a chromatographic column with calcium dihydrogen orthophosphate monohydrate, treating said column with sufficient alkali to adjust its pH to the range of from about 5.5 to about 8.0 at a temperature of not more than about 40* C., said column thereafter being washed with sufficient water to raise the pH of the column at least about 0.2 units to the range of about 5.7 to about 8.2, further washing said column with an alkali metal trimetaphosphate solution having a pH of about 8, flowing unto the column a diluted virus solution, said virus solution having been treated with an alkali metal trimetaphosphate and having a pH of about 8.0 and eluting said column with a phosphate buffer having a pH of about 7.0.
 3. A method as described in claim 1 for purifying influenza virus which comprises packing a chromatographic column with calcium dihydrogen orthophosphate monohydrate, treating said column with sufficient alkali to adjust its pH from about 5.5 to about 8.0 at a temperature of not more than about 40* C., said column thereafter being washed with sufficient distilled water to provide the column with a pH of about 7.5, further washing said column with a solution of an alkali metal trimetaphosphate, flowing unto the column a diluted influenza virus containing fluid, said virus having been treated with an alkali metal trimetaphosphate so as to have a pH of about 8.0 and eluting the influenza virus from said column with a phosphate buffer having a molarity of about 0.3 to about 1.0 and a pH of about 7.0.
 4. A method as described in claim 1 for purifying influenza virus which comprises packing a chromatographic column with calcium dihydrogen orthophosphate monohydrate, treating said column at about room temperature with sufficient alkali to adjust the pH to about 6.5, said column thereafter being washed with sufficient distilled water to provide the column with a pH of about 7.5, further washing said column with a sodium trimetaphosphate solution having a pH of about 8, flowing unto the column a dilute influenza virus containing fluid, said virus having been treated with sodium trimetaphosphate so as to have a pH of about 8.0 and eluting the influenza virus from said column with a 1.0 molar phosphate buffer having a pH of about 7.0.
 5. A method for purifying virus which comprises packing a chromatographic column with calcium dihydrogen orthophosphaTe monohydrate, treating said column at a temperature of not more than about 40* C. with sufficient alkali to adjust its pH to the range of from about 5.5 to about 8.0, said column thereafter being washed with sufficient water to raise the pH of the column at least about 0.2 units to the range of about 5.7 to about 8.2, further washing said column with a solution of an alkali metal trimetaphosphate, flowing unto the column an influenza virus containing fluid and eluting said column with a phosphate buffer having a pH of about 7.0. 